KRT1 gene silencing ameliorates myocardial ischemia–reperfusion injury via the activation of the Notch signaling pathway in mouse models

Myocardial ischemia and reperfusion injury (MIRI) includes major drawbacks, such as excessive formation of free radicals and also overload of calcium, which lead to cell death, tissue scarring, and remodeling. The current study aims to explore whether KRT1 silencing may ameliorate MIRI via the Notch signaling pathway in mouse models. Myocardial tissues were used for the determination of the positive rate of KRT1 protein expression, apoptosis of myocardial cells, creatine kinase (CK) and lactate dehydrogenase (LDH) expression, expression of related biomarkers as well as myocardial infarction area. The transfected myocardial cells were treated with KRT1-siRNA, Jagged1, and DAPT (inhibitor of Notch-1 signaling pathway). The expression of KRT1, NICD, Hes1, Bcl-2, and Bax protein was detected. The MTT assay was applied for cell proliferation and flow cytometry was used for cell apoptosis. Mice with MIRI had a higher positive rate of KRT1 protein expression, apoptosis of myocardial cells, CK and LDH expression, myocardial infarction area, increased expression of MDA, NO, SDH, IL-1, IL-6, TNF-α, CRP, KRT1, Bax protein, CK, and LDH, and decreased expression of SOD, NICD, Hes1, and Bcl-2. The downregulation of KRT1 led to decreased expression of KRT1 and Bax protein, increased expression of NICD, Hes1, and Bcl-2, decreased cell apoptosis, and improved cell proliferation. The inhibition of the Notch signaling pathway leads to reduced expression of Bax, increased expression of NICD, Hes1, and Bcl 2, and also decreased cell apoptosis and increased cell proliferation. Our data conclude that KRT1 silencing is able to make MIRI better by activating the Notch signaling pathway in mice.     

Caveolin-1 promote astroglial differentiation of neural stem/progenitor cells through modulating Notch1/NICD and Hes1 expressions

In the present study, we aim to elucidate the role of caveolin-1 in modulating astroglial differentiation of neural progenitor cells (NPCs) and the potential mechanisms involved. We first investigated astroglial differentiation and Notch signaling by detecting the expressions of S100β, GFAP, NICD and hairy enhancer of split 1 (Hes1) in the brains of wild-type and caveolin-1 knockout mice. Caveolin-1 knockout mice revealed remarkably less astroglial differentiation and lower levels of NICD and Hes1 expressions than wild type mice. We then studied the potential roles of caveolin-1 in modulating NICD and Hes1 expressions and astroglial differentiation in isolated cultured NPCs by using caveolin-1 peptide and caveolin-1 RNA silencing. In the differentiating NPCs, caveolin-1 peptide markedly promoted astroglial formation and up-regulated the expressions of NICD and Hes1. In contrast, the knockdown of caveolin-1 inhibited astroglial differentiation of NPCs and the expressions of NICD and Hes1. Taken together, these results provide strong evidence that caveolin-1 can promote astroglial differentiation of NPCs through modulating Notch1/NICD and Hes1 expressions.     

LncRNA SNHG1 promotes sepsis‐induced myocardial injury by inhibiting Bcl‐2 expression via DNMT1

Myocardial injury is a frequently occurring complication of sepsis. This study aims to investigate the molecular mechanism of long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1)‐mediated DNA methyltransferase 1/B‐cell lymphoma‐2 (DNMT1/Bcl‐2) axis in sepsis‐induced myocardial injury. Mice and HL‐1 cells were treated with lipopolysaccharide (LPS) to establish animal and cellular models simulating sepsis and inflammation. LncRNA SNHG1 was screened out as a differentially expressed lncRNA in sepsis samples through microarray profiling, and the upregulated expression of lncRNA SNHG1 was confirmed in myocardial tissues of LPS‐induced septic mice and HL‐1 cells. Further experiments suggested that silencing of lncRNA SNHG1 reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. LncRNA SNHG1 inhibited Bcl‐2 expression by recruiting DNMT1 to Bcl‐2 promoter region to cause methylation. Inhibition of Bcl‐2 promoter methylation reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. In vivo experiments substantiated that lncRNA SNHG1 silencing alleviated sepsis‐induced myocardial injury in mice. Taken together, lncRNA SNHG1 promotes LPS‐induced myocardial injury in septic mice by downregulating Bcl‐2 through DNMT1‐mediated Bcl‐2 methylation.     

Notch Signaling in Descending Thoracic Aortic Aneurysm and Dissection

Background

Descending thoracic aortic aneurysm and dissection (DTAAD) is characterized by progressive medial degeneration, which may result from excessive tissue destruction and insufficient repair. Resistance to tissue destruction and aortic self-repair are critical in preventing medial degeneration. The signaling pathways that control these processes in DTAAD are poorly understood. Because Notch signaling is a critical pathway for cell survival, proliferation, and tissue repair, we examined its activation in DTAAD.

Methods

We studied descending thoracic aortic tissue from patients with sporadic thoracic aortic aneurysm (TAA; n = 14) or chronic thoracic aortic dissection (TAD; n = 16) and from age-matched organ donors (n = 12). Using western blot, real-time RT-PCR, and immunofluorescence staining, we examined aortic tissue samples for the Notch ligands Delta-like 1, Delta-like 4 (DLL1/4), and Jagged1; the Notch receptor 1 (Notch1); the Notch1 intracellular domain (NICD); and Hes1, a downstream target of Notch signaling.

Results

Western blots and RT-PCR showed higher levels of the Notch1 protein and mRNA and the NICD and Hes1 proteins in both TAA and TAD tissues than in control tissue. However, immunofluorescence staining showed a complex pattern of Notch signaling in the diseased tissue. The ligand DLL1/4 and Notch1 were significantly decreased and NICD and Hes1 were rarely detected in medial vascular smooth muscle cells (VSMCs) in both TAA and TAD tissues, indicating downregulation of Notch signaling in aortic VSMCs. Interestingly Jagged1, NICD, and Hes1 were highly present in CD34+ stem cells and Stro-1+ stem cells in aortas from TAA and TAD patients. NICD and Hes1 were also detected in most fibroblasts and macrophages that accumulated in the aortic wall of DTAAD patients.

Conclusions

Notch signaling exhibits a complex pattern in DTAAD. The Notch pathway is impaired in medial VSMCs but activated in stem cells, fibroblasts, and macrophages.     

The anti‐apoptotic Bcl‐2 protein regulates hair follicle stem cell function

Maintaining the architecture, size and composition of an intact stem cell (SC) compartment is crucial for tissue homeostasis and regeneration throughout life. In mammalian skin, elevated expression of the anti‐apoptotic Bcl‐2 protein has been reported in hair follicle (HF) bulge SCs (BSCs), but its impact on SC function is unknown. Here, we show that systemic exposure of mice to the Bcl‐2 antagonist ABT‐199/venetoclax leads to the selective loss of suprabasal BSCs (sbBSCs), thereby disrupting cyclic HF regeneration. RNAseq analysis shows that the pro‐apoptotic BH3‐only proteins BIM and Bmf are upregulated in sbBSCs, explaining their addiction to Bcl‐2 and the marked susceptibility to Bcl‐2 antagonism. In line with these observations, conditional knockout of Bcl‐2 in mouse epidermis elevates apoptosis in BSCs. In contrast, ectopic Bcl‐2 expression blocks apoptosis during HF regression, resulting in the accumulation of quiescent SCs and delaying HF growth in mice. Strikingly, Bcl‐2‐induced changes in size and composition of the HF bulge accelerate tumour formation. Our study identifies a niche‐instructive mechanism of Bcl‐2‐regulated apoptosis response that is required for SC homeostasis and tissue regeneration, and may suppress carcinogenesis.     

Decreased p53 is associated with a decline in asymmetric stem cell self‐renewal in aged human epidermis

With age, the epidermis becomes hypoplastic and hypoproliferative. Hypoproliferation due to aging has been associated with decreased stem cell (SC) self‐renewal in multiple murine tissues. The fate of SC self‐renewal divisions can be asymmetric (one SC, one committed progenitor) or symmetric (two SCs). Increased asymmetric SC self‐renewal has been observed in inflammatory‐mediated hyperproliferation, while increased symmetric SC self‐renewal has been observed in cancers. We analyzed SC self‐renewal divisions in aging human epidermis to better understand the role of SCs in the hypoproliferation of aging. In human subjects, neonatal to 78 years, there was an age‐dependent decrease in epidermal basal layer divisions. The balance of SC self‐renewal shifted toward symmetric SC self‐renewal, with a decline in asymmetric SC self‐renewal. Asymmetric SC divisions maintain epidermal stratification, and this decrease may contribute to the hypoplasia of aging skin. P53 decreases in multiple tissues with age, and p53 has been shown to promote asymmetric SC self‐renewal. Fewer aged than adult ALDH+CD44+ keratinocyte SCs exhibited p53 expression and activity and Nutlin‐3 (a p53 activator) returned p53 activity as well as asymmetric SC self‐renewal divisions to adult levels. Nutlin‐3 increased Notch signaling (NICD, Hes1) and DAPT inhibition of Notch activation prevented Nutlin‐3 (p53)‐induced asymmetric SC self‐renewal divisions in aged keratinocytes. These studies indicate a role for p53 in the decreased asymmetric SC divisions with age and suggest that in aged keratinocytes, Notch is required for p53‐induced asymmetric SC divisions.     

Bcl‐xL expression following articular cartilage injury and its effects on the biological function of chondrocytes

This study aimed to investigate the expression of B‐cell lymphoma‐extra large (Bcl‐xL) in cartilage tissues following articular cartilage injury and to determine its effects on the biological function of chondrocytes. A total of 25 necrotic cartilage tissue samples and 25 normal tissue samples were collected from patients diagnosed with osteoarthritis at our hospital from December 2015 to December 2018. The mRNA expression levels of Bcl‐xL, caspase‐3, and matrix metalloproteinase‐3 (MMP‐3) in the normal and necrotic tissues were examined via quantitative polymerase chain reaction, and their protein expression levels were detected via western blotting. The expression levels of Bcl‐xL, insulin‐like growth factor‐1 (IGF‐1), and bone morphogenetic protein (BMP) were significantly lower but those of caspase‐3, MMP‐3, interleukin‐1β (IL‐1β), and chemokine‐like factor 1 (CKLF1) levels were markedly higher in necrotic cartilage tissues than in normal tissues. Following cell transfection, the expression levels of Bcl‐xL, IGF‐1, and BMP were remarkably higher but those of caspase‐3, MMP‐3, IL‐1β, and CKLF1 were notably lower in the Si‐Bcl‐xL group than in the NC group. The Si‐Bcl‐xL group showed significantly lower cell growth and noticeably higher apoptosis rate than the NC group (normal control group). The expression of Bcl‐xL is reduced following articular cartilage injury, and this reduction promotes the proliferation and inhibits the apoptosis of chondrocytes. Therefore, Bcl‐xL could serve as a relevant molecular target in the clinical practice of osteoarthritis and other diseases causing cartilage damage.     

CircSAMD4A aggravates H/R‐induced cardiomyocyte apoptosis and inflammatory response by sponging miR‐138‐5p

Hypoxia/reoxygenation (H/R)‐induced myocardial cell injury is the main cause of acute myocardial infarction (AMI). Many proofs show that circular RNA plays an important role in the development of AMI. The purpose of this study was to investigate the role of circSAMD4A in H/R‐induced myocardial injury. The levels of circular SAMD4A (circSAMD4A) were detected in the heart tissues of AMI mice and H/R‐induced H9C2 cells, and the circSAMD4A was suppressed in AMI mice and H/R‐induced H9C2 cells to investigate its’ function in AMI. The levels of circSAMD4A and miR‐138‐5p were detected by real‐time quantitative PCR, and MTT assay was used to detect cell viability. TUNEL analysis and Annexin V‐FITC were used to determine apoptosis. The expression of Bcl‐2 and Bax proteins was detected by Western blot. IL‐1β, TNF‐α and IL‐6 were detected by ELISA kits. The study found that the levels of circSAMD4A were up‐regulated after H/R induction and inhibition of circSAMD4A expression would reduce the H/R‐induced apoptosis and inflammation. MiR‐138‐5p was down‐regulated in H/R‐induced H9C2 cells. circSAMD4A was a targeted regulator of miR‐138‐5p. CircSAMD4A inhibited the expression of miR‐138‐5p to promote H/R‐induced myocardial cell injury in vitro and vivo. In conclusion, CircSAMD4A can sponge miR‐138‐5p to promote H/R‐induced apoptosis and inflammatory response.     

Enhanced effect of recombinant adenoviruses co‐expression of ING4 and OSM on anti‐tumour activity of laryngeal cancer

The inhibitor of growth family member 4 (ING4) is one of the ING family genes, serves as a repressor of angiogenesis or tumour growth and suppresses loss of contact inhibition. Oncostatin M (OSM) is a multifunctional cytokine that belongs to the interleukin (IL)‐6 subfamily with several biological activities. However, the role of recombinant adenoviruses co‐expressing ING4 and OSM (Ad‐ING4‐OSM) in anti‐tumour activity of laryngeal cancer has not yet been identified. Recombinant Ad‐ING4‐OSM was used to evaluate their combined effect on enhanced anti‐tumour activity in Hep‐2 cells of laryngeal cancer in vivo. Moreover, in vitro function assays of co‐expression of Ad‐ING4‐OSM were performed to explore impact of co‐expression of Ad‐ING4‐OSM on biological phenotype of laryngeal cancer cell line, that is Hep‐2 cells. In vitro, Ad‐ING4‐OSM significantly inhibited the growth, enhanced apoptosis, altered cell cycle with G1 and G2/M phase arrest, and upregulated the expression of P21, P27, P53 and downregulated survivin in laryngeal cancer Hep‐2 cells. Furthermore, in vivo functional experiments of co‐expressing of Ad‐ING4‐OSM demonstrated that solid tumours in the nude mouse model were significantly suppressed, and the co‐expressing Ad‐ING4‐OSM showed a significant upregulation expression of P21, P53, Bax and Caspase‐3 and a downregulation of Cox‐2, Bcl‐2 and CD34. This study for the first time demonstrated the clinical value and the role of co‐expressing Ad‐ING4‐OSM in biological function of laryngeal cancer. This work suggested that co‐expressing Ad‐ING4‐OSM might serve as a potential therapeutic target for laryngeal cancer patients.     

The Notch γ-secretase inhibitor ameliorates kidney fibrosis via inhibition of TGF-β/Smad2/3 signaling pathway activation

Kidney fibrosis is a common feature of chronic kidney disease (CKD). A recent study suggests that abnormal Notch signaling activation contributes to the development of renal fibrosis. However, the molecular mechanism that regulates this process remains unexplored. Unilateral ureteral obstruction (UUO) or sham-operated C57BL6 mice (aged 10 weeks) were randomly assigned to receive dibenzazepine (DBZ, 250 μg/100 g/d) or vehicle for 7 days. Histologic examinations were performed on the kidneys using Masson's trichrome staining and immunohistochemistry. Real-time PCR and western blot analysis were used for detection of mRNA expression and protein phosphorylation. The expression of Notch 1, 3, and 4, Notch intracellular domain (NICD), and its target genes Hes1 and HeyL were upregulated in UUO mice, while the increase in NICD protein was significantly attenuated by DBZ. After 7 days, the severity of renal fibrosis and expression of fibrotic markers, including collagen 1α1/3α1, fibronectin, and α-smooth muscle actin, were markedly increased in UUO compared with sham mice. In contrast, administration of DBZ markedly attenuated these effects. Furthermore, DBZ significantly inhibited UUO-induced expression of transforming growth factor (TGF)-β, phosphorylated Smad 2, and Smad 3. Mechanistically, Notch signaling activation in tubular epithelial cells enhanced fibroblast proliferation and activation in a coculture experiment. Our study provides evidence that Notch signaling is implicated in renal fibrogenesis. The Notch inhibitor DBZ can ameliorate this process via inhibition of the TGF-β/Smad2/3 signaling pathway, and might be a novel drug for preventing chronic kidney disease.     

Mind bomb1 is a ubiquitin ligase essential for mouse embryonic development and Notch signaling

The Notch-Delta signaling pathway controls many conserved cell determination events. While the Notch end is fairly well characterized, the Delta end remains poorly understood. Mind bomb1 (MIB1) is one of two E3 ligases known to ubiquitinate Delta. We report here that a targeted mutation of Mib1 in mice results in embryonic lethality by E10.5. Mutants exhibit multiple defects due to their inability to modulate Notch signaling. As histopathology revealed a strong neurogenic phenotype, this study concentrates on characterizing the Mib1 mutant by analyzing Notch pathway components in embryonic neuroepithelium prior to developmental arrest. Premature neurons were observed to undergo apoptosis soon after differentiation. Aberrant neurogenesis is a direct consequence of lowered Hes1 and Hes5 expression resulting from the inability to generate Notch1 intracellular domain (NICD1). We conclude that MIB1 activity is required for S3 cleavage of the Notch1 receptor. These results have direct implications for manipulating the differentiation of neuronal stem cells and provide a putative target for the modulation of specific tumors.     

Membrane‐dependent amyloid aggregation of human BAX α9 (173–192)

Mitochondrial outer membrane permeabilization, which is a critical step in apoptosis, is initiated upon transmembrane insertion of the C‐terminal α‐helix (α9) of the proapoptotic Bcl‐2 family protein BAX. The isolated α9 fragment (residues 173–192) is also competent to disrupt model membranes, and the structures of its membrane‐associated oligomers are of interest in understanding the potential roles of this sequence in apoptosis. Here, we used ultrafast two‐dimensional infrared (2D IR) spectroscopy, thioflavin T binding, and transmission electron microscopy to show that the synthetic BAX α9 peptide (α9p) forms amyloid aggregates in aqueous environments and on the surfaces of anionic small unilamellar vesicles. Its inherent amyloidogenicity was predicted by sequence analysis, and 2D IR spectra reveal that vesicles modulate the β‐sheet structures of insoluble aggregates, motivating further examination of the formation or suppression of BAX amyloids in apoptosis.